Driven by recent technical advances in genetic engineering and new societal needs, the use of enzymes and microorganisms as catalysts to synthesize chemicals and materials is rapidly expanding. However, many challenges have yet to be fully addressed, such as developmental costs of biocatalysts and the type of chemistry performed. Most biocatalysts currently used in industry (˜65%) are hydrolases that do not perform complex chemistry. The primary reason for this lack of use of complicated chemical reactions is that enzymes catalyzing more involved transformations often require one or more costly cofactors, making these reactions industrially impractical when the cofactor is added in a stoichiometric amount.
Oxidoreductases, for example, can be used for synthesis of chiral compounds, complex carbohydrates, and isotopically labeled compounds, but they often require NADH or NADPH as cofactors. The cost of NADH is about $40/mmol, whereas the price of NADPH is nearly $500/mmol (Sigma 2002 catalog), rendering stoichiometric use of either reduced cofactor at the kilogram scale prohibitively expensive. There is a need, therefore, to develop regeneration systems for NAD(P)(H) that would allow their addition in catalytic amounts, with the goal of making redox bioprocesses industrially feasible. Because approximately 80% of all reductases utilize NAD(P)(H) as a cofactor, probably accounting for over 300 known reactions, regeneration of these cofactors would be particularly advantageous.
A number of enzymatic, electrochemical, chemical, photochemical, and biological methods have been developed to regenerate cofactors. Advantages of cofactor regeneration in addition to reduced costs include simplified reaction work up, prevention of product inhibition from the cofactor, and sometimes a favorable influence on the reaction equilibrium. In some uses, the regenerative system drives the synthetic reaction forward, even when the formation of the desired product is less favored under standard conditions. Specific advantages of enzymatic strategies include high selectivity, compatibility with synthetic enzymes, and high turnover numbers. Aspects to be considered when using enzymatic methods include the expense and stability of the enzyme, cost of the substrate for the regenerative enzyme, ease of product purification, catalytic efficiency, KM for the cofactor, and thermodynamic driving force of the regenerative enzyme.
Of the enzymatic NADH regeneration systems, the best and most widely used enzyme is formate dehydrogenase (FDH) from Candida boidini. Phosphite dehydrogenase (PTDH) may have kinetic and practical advantages over FDH in certain applications, e.g. using PTDH as a regeneration system. This enzyme catalyzes the nearly irreversible oxidation of hydrogen phosphonate (phosphite) to phosphate, with the concomitant reduction of NAD+ to NADH. The large change in free energy of this reaction (ΔG°=−63.3 kJ/mol estimated from redox potentials) and the associated high equilibrium constant (Keq=1×1011) makes PTDH a promising NADH regenerative enzyme. A particularly interesting application of PTDH is the facile production of isotopically labeled products. Deuterium or tritium labeled water can be used to readily and economically prepare labeled phosphite. Subsequent use of isotopically labeled phosphite during a synthetic reduction using PTDH for NADH regeneration has been shown to efficiently generate labeled products in high isotopic purity.
NADPH is significantly more expensive than NADH and currently no widely used system for its regeneration is available. The most promising enzymatic NADPH regeneration system is a mutant FDH from Pseudomonas sp. 101 (mut-Pse FDH) available from Jülich Fine Chemicals. However, the enzyme's mutations have not been made public, the catalytic efficiency is low (1 μM min−1), and the cost is high. Another alternative is the use of a soluble pyridine nucleotide transhydrogenase which catalyses the transfer of reducing equivalents between NAD+ and NADP+. Unfortunately, this route would require addition of both cofactors and a third enzyme to the process. Currently, the high cost of regenerating enzymes and inefficient regeneration makes the production of synthetic products requiring the use of NADPH not very attractive.
There are reports about the alteration of nicotinamide cofactor specificity including determinants and evolution of nicotinamide binding sites. However, altering cofactor specificity remains a challenge, because very few examples exist where catalytic efficiency for the disfavored cofactor NADPH has been significantly improved to approximately the activity with the preferred substrate. Even fewer are the examples where specificity becomes relaxed allowing high catalytic efficiency with both NAD(H) and NADP(H). Among this last group are the non-Rossman fold NAD+-dependent isocitrate dehydrogenase, glucose-fructose oxidoreductase, glutathione reductase, and aldehyde dehydrogenase. A comparison of the strategies required to achieve efficient use of the non-physiological cofactor in these enzymes indicates that there is no clear recipe for success.